LOCUS       sequence-484722-        6193 bp    DNA     circular SYN 27-AUG-2025
DEFINITION  Part 2 of a two-plasmid PCA biosensor composed of the PcaV 
            repressible PPV promoter upstream from a reporter gene (eGFP) on a 
            second plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6193)
  AUTHORS   F M Machado L, Currin A, Dixon N
  TITLE     Directed evolution of the PcaV allosteric transcription factor to 
            generate a biosensor for aromatic aldehydes.
  JOURNAL   J Biol Eng. 2019 Nov 27;13:91. doi: 10.1186/s13036-019-0214-z. 
            eCollection 2019.
  PUBMED    31798685
REFERENCE   2  (bases 1 to 6193)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 27, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Eng."; date: "2019-11-27"; volume: "13:91. doi"; pages: " 
            10.1186/s13036-019-0214-z. eCollection 2019"
COMMENT     Sequence Label: p44PPV-eGFP
FEATURES             Location/Qualifiers
     source          1..6193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..115
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(367..1158,1159..1227))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     990..1009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1228..1320)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1401..1989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1890..1909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2143..2160
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2175..2314
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2240..2259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2400..2422
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2416..2607)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3179..4261)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4223..4242
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4262..4339)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4546..4563
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4573..4590
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(4594..4596,4597..4599,4600..5313)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4594..4596,4597..4599,4600..5310)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4639..4660)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4900..4919)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5247..5268
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5411..5443
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             5450..5467
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5633..5719
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5811..5838
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5892..5910)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5906..5953
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5990..6193,1..252)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6077..6096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
       61 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      121 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      181 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      241 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
      301 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
      361 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      421 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      481 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
      541 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      601 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
      661 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
      721 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
      781 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
      841 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
      901 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
      961 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     1021 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     1081 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     1141 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     1201 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     1261 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     1321 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1381 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1441 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1501 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     1561 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1621 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1681 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     1741 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     1801 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     1861 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     1921 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     1981 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2041 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2101 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2161 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2221 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     2281 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2341 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2401 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     2461 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     2521 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     2581 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     2641 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     2701 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     2761 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     2821 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     2881 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     2941 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3001 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3061 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     3121 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     3181 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3241 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     3301 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     3361 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     3421 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     3481 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     3541 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     3601 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     3661 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     3721 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     3781 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     3841 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     3901 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     3961 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     4021 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     4081 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     4141 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     4201 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     4261 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     4321 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     4381 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     4441 aaaatttatc aaaaagagtg ttgactatac tcagtgccct gactatgata cttagattca
     4501 tactcagtgc cctgactatg aaggagatat accatgggca gcagccatca tcatcatcat
     4561 cacagcagcg gcctggtgcc gcgcggcagc catatggtga gcaagggcga ggagctgttc
     4621 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     4681 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     4741 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     4801 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     4861 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     4921 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     4981 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5041 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5101 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5161 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     5221 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5281 atcactctcg gcatggacga gctgtacaag taaggatccg aattctgtac aggcgcgcct
     5341 gcaggacgtc gacggtacca tcgatacgcg ttcgaagctt gcggccgcac agctgtatac
     5401 acgtgcaagc cagccagaac tcgctcctga agacccagag gatctcgagc accaccacca
     5461 ccaccactaa tgttaattaa gttgggcgtt cctaggctga taaaacagaa tttgcctggc
     5521 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
     5581 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa
     5641 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     5701 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg
     5761 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat
     5821 cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt
     5881 caaatatgta tccgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     5941 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     6001 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6061 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6181 cacctcgacc cca
//
